Pier Giorgio Pifferi

The stabilization of white wines by adsorption of phenolic compounds on chitin and chitosan

Browning and over-oxidation are the most common alterations affecting white wines. The reduction of phenolic compounds by means of adsorbents is the method most frequently employed in oenology for countering these phenomena. The purpose of this study was to investigate the use of two types of adsorbents not as yet employed in oenology, chitin and chitosan, on a variety of white wines (Sauvignon, Pinot, Trebbiano, Albana, Verdicchio, Bianco di Custoza) and to compare their behaviour with that of two conventional adsorbents already used for these applications, potassium caseinate and polyvinyl pyrrolidone. The chitosans exhibited a high affinity to phenolic compounds (polyphenols, cinnamic acids, flavans, proanthocyanidins), in particular to cinnamic acids, and good stabilization to browning over time as determined by an accelerated test. Two of the chitosans tested were found to be comparable to the conventional adsorbents.

Immobilization of β-glucosidase from a commercial preparation. Part 2. Optimization of the immobilization process on chitosan

β-Glucosidase was adsorbed on chitosan and crosslinked with glutaraldehyde. The enzyme exhibited a considerable affinity to chitosan, giving good immobilization yields (55–85%) while maintaining an optimum level of activity (550–850 U/g). The stability in acid buffer (T12>80–100 days) and in wine (T12>55 days) was also high. The results are promising for a future technological application of the immobilized enzyme in wine-making.

Kinetic and immobilization studies on fungal glycosidases for aroma enhancement in wine

Abstract The biochemical properties of a commercial glycosidase from Aspergillus niger (Cytolase PCL5, Genencor) were investigated. The product contains β-glucosidase, α-arabinosidase, and α-rhamnosidase activities in a ratio considered suitable for aroma enhancement in wine-making. A kinetic study of these three activities was carried out, which included determination of kinetic constants; dependence of enzyme activity and stability on pH and temperature; and enzyme inhibition by glucose, fructose, glycerol, and sulfurous anhydride. These glycosidase activities were immobilized to a solid carrier with the aim of developing a continuous process for wine aroma enhancement. Immobilization was best achieved with silanized bentonite as the solid, activated carrier, with bound glutaraldehyde as the reactive arm at a protein:carrier ratio of 0.11. Immobilization reaction was carried out for 48 h at pH 4 and 30°C. Under these conditions, 16 units of β-glucosidase, 2 units of α-arabinosidase, and 4.5 units of α-rhamnosidase per gram of bentonite were immobilized.

A simple method for purifying glycosidases: α-l-rhamnopyranosidase from Aspergillus niger to increase the aroma of Moscato wine

alpha-L-rhamnopyranosidase (Rha, EC 3.2.1.40) is an enzyme of considerable importance in food technology for increasing the aroma of wines, musts, fruit juices and other alcoholic beverages. The aim of this research is to study the purification of Rha contained in a commercial preparation already used in the winemaking industry. With the procedure adopted, Rha recovery values were excellent (ca 85%), comparable with those we found in a previous paper on the purification of other glycosidases such beta-D-glucopyranosidase (betaG) and alpha-L-arabinofuranosidase (Ara) [1]. The Rha purification value (4.3) and drastic reduction in brown compounds (DeltaAbs 95%) represent other strengths of the proposed method that has proved inexpensive and simple to apply. In addition, purified Rha has shown itself to be more stable than other glycosidases. This had optimum effect at pH 4, while optimum temperature was 70 degrees C, greater than that found for other glycosidases. The purified enzyme was characterized in terms of the kinetic parameters K(m) (1.40 mM) and V(max) (1.30 U mg(-1) of protein) and subsequently used to increase aroma a model wine solution containing aromatic precursors extracted from the skins of Moscato grapes, with an increase in the content of total terpenols of ca 2.3 times.

Laccase catalyzed-oxidative coupling of 3-methyl 2-benzothiazolinone hydrazone and methoxyphenols

Abstract The reaction between o -, m -, and p -methoxyphenols and 3-methyl-2-benzothiazolinone hydrazone was studied in the presence of laccase from Pyricularia oryzae . The findings show that laccase (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) catalyzes the oxidative coupling reaction between MBTH and phenols producing red colored azo-dye compounds. On the basis of kinetic parameters and optimum pH values, the mechanisms of the oxidative coupling reactions with the different phenols are discussed. The results suggest that the reaction is initialized by the enzymatic 3-methyl-2-benzothiazolinone hydrazone activation, which undergoes electrophilic substitution with m -methoxyphenol in solution, enzymatic activated guaiacol, and chelated p -methoxyphenol at the catalytic site of the laccase. The current study also demonstrates the possibility of using the azo-dye formed in the presence of guaiacol for measuring laccase activity following a similar assaying method as that used for measuring peroxidase activity. This type of assay permits the determination of laccase at micromolar levels by fixed time method.

A simple Method for Purifying Glycosidases: α-l-arabinofuranosidase and β-d-glucopyranosidase from Aspergillus niger to Increase the Aroma of Wine. Part I

Abstract The purpose of this paper was to study the purification of two glycosidases, α- l -arabinofuranosidase (EC 3.2.1.55) and β- d -glucopyranosidase (EC 3.2.1.21), contained in a commercial preparation. The final purification procedure entailed precipitation in ethanol, ultrafiltration, adsorption on bentonite, salting with KCl, and ultrafiltration. The procedure is both simple and inexpensive to perform at an industrial level. It allows the recovery of 70% of α- l -arabinofuranosidase and 116% of β- d -glucopyranosidase activities with purification values of 10.4 and 6.4, respectively. The amounts of brown compounds and polysaccharides are also considerably reduced by about 80%. The enzymes thus purified were subsequently characterized in terms of optimum pH and temperature, stability over time, kinetic parameters ( K m and V max ), and then employed for aromatizing a model wine solution containing aromatic precursors extracted from Moscato grape skin.

Immobilization of a pectinlyase from Aspergillus niger for application in food technology

Abstract The immobilization of a commercial preparation of pectinlyase (PL, EC 4.2.2.3) derived from Aspergillus niger was studied in view of its possible application in fruit juice treatments. Pectinlyase is an enzyme that is subject to growing interest for the substitution of other pectic enzymes: e.g., polygalacturonase (EC 3.2.1.15) and pectinesterase (EC 3.1.1.11). The PL was immobilized by physical adsorption or by the formation of covalent bonds on organic (cellulose and its derivatives, XAD-amberlites) and inorganic (sulphides, γ-alumina, and bentonite) supports. The supports that permitted an effective immobilization with good activity levels and sufficient stability under the operational conditions were found to be the acrylic resin XAD 7 activated with trichlorotriazine and, in particular, bentonite activated with glutaraldehyde.

On the use of chitosan-immobilized β-glucosidase in wine-making: kinetics and enzyme inhibition

Abstract The kinetics of chitosan-immobilized β-glucosidase and enzyme inhibition by several components of wine and must (glucose, fructose and terpenols) were studied. Optimum immobilization conditions were: temperature 25°C, pH between 5·5 and 6·0, polymeric support dimension in the range 38–75 μm, cross-linking time 30 min, glutaraldehyde concentration 0·5–1·0% w/v, 1 g of chitosan per 1000 units of β-glucosidase. The immobilized enzyme retained 29% of the wet biocatalyst activity when freeze-dried and showed good stability (half-life roughly 2 years) when stored at 4°C. Kinetics were tested at 25°C following the hydrolysis of p-nitrophenyl β- d glucopyranoside and obey the Michaelis-Menten rate equation. Km = 1·3 mM and the activation energy, 62·84 kJ mol−1, are close to those of the free enzyme. The operational half-life was roughly 500 h.Glucose only depressed the enzyme activity according to a reversible non-competitive inhibition mechanism with Ki = 11·2 mM.

Immobilization of β-glucosidase from a commercial preparation. Part 1. A comparative study of natural supports

Abstract A number of natural supports (cellulose PEI, alpha-alumina, gamma-alumina and chitosan) were tested for the immobilization of β-glucosidase in view of its possible application in the wine-making and fruit-juice processing industry. Immobilization on chitosan produced an active catalyst (about 500 U/g) and a shift of pH optimum to lower values, which is of considerable interest for use in the beverage processing industry. However, the short storage life of the immobilized enzyme is currently a drawback to its industrial applications.

A novel chitosan derivative to immobilize α-L-rhamnopyranosidase from Aspergillus niger for application in beverage technologies

alpha-L-rhamnopyranosidase (Rha, EC 3.2.1.40) is an enzyme of considerable importance to food technology in increasing the aroma of wines, musts, fruit juices and other beverages. The aim of this research is the immobilization of the Rha contained in a commercial preparation already used in the winemaking industry and purified in the manner described in a previous study [1]. The immobilization supports tested were chitin, chitosan and derivatized chitosan, diethylaminoethyl chitosan (DE-chitosan) never previously used for this type of application. Particularly, on DE-chitosan, the Rha was adsorbed and cross-linked with various bifunctional agents (glutaraldehyde, diepoxyoctane, suberimidate and carbodiimide), whose best results (immobilization yields and activity) were obtained with carbodiimide (EDC) that allowed a reduction in the involvement of the enzyme amine groups that are probably important in catalytic mechanism. In addition, the use of rhamnose and a succinimide (NHS) during cross-linking enhanced the action of the EDC and so increased the immobilization yield and activity. The immobilized Rha retained the kinetic parameters (K(m) and V(max)) of the free enzyme and increased stability. Moreover, this biocatalyst allowed an increase in the aroma in a model wine solution containing glicosidic precursors with a marked reduction in specificity toward tertiary monoterpenols as compared to the free enzyme.